The present invention relates to phosphorus-32 and phosphorus-33 labeled proteins which are useful for radiotherapy of human diseases. In particular, the invention relates to proteins which contain peptide sequences that are substrates for protein kinase enzymes, and that can be radiolabeled with a protein kinase and a 32P or 33P-labeled phosphate donor and contain an SH2 domain which serves to protect the phosphorylated protein from in vivo dephosphorylation. This invention also relates to a method of therapy using the radiolabeled proteins.
Many radionuclides have been studied for their suitability for internal administration to patients in radiotherapy. Some radionuclide compounds, containing isotopes such as 131I, can be given systemically, taking advantage of the fact that these elements tend to localize to particular tissues by virtue of their chemical properties. Other radionuclides, such as 198Au and 103Pd have been administered in a localized fashion, for instance to the site of a tumor. Most recent approaches, however, have focused on methods of delivering radionuclides to a preselected tissue by attaching the radionuclide to a targeting protein, usually an antibody, which will then localize to that tissue.
A large number of methods for associating radionuclides to antibodies have been developed. The chemical toxicity of many radionuclides means that complex methods must often be used to stably bind the isotope to an antibody. For example, to use 90Y, which has many desirable radiochemical properties, a chelate must be synthesized and covalently bound to the antibody to stably link the radioisotope to the antibody.
Currently there are isotopes which have been utilized in radiolabeled agents and include 131I, 90y, 188Re, 186Re, 67Cu, and 212Pb/212Bi. Each has significant, sometimes multiple, disadvantages including low-energy particle emissions, such as 131I and 67CU; severe organ toxicity, such as 90Y and 212Pb/212Bi; short half-life for radioimmunotherapy, such as 188Re and 212Pb/212Bi; high gamma-energy emissions, such as 131I; low specific activity, such as 186Re; non-amenability to out-patient procedures, such as 131I, 67Cu and 212Pb/212Bi and supply and/or cost concerns, such as 188Re, 186Re, 67Cu, and 212Pb/212Bi.
One isotope which has received little attention, due to the difficulty of the chemistry involved in linking it to antibodies, but otherwise displays desirable properties for radioimmunotherapy is 32P. 32P is inexpensive, is readily available in high specific activity in a variety of labeled molecules, and has a therapeutically desirable half-life of 14 days. Additionally, it has been previously used clinically, has no gamma emissions, is carrier-free and has an intense beta-emission. It is absorbed by the body and is not readily excreted, and is therefore amenable to use in outpatient procedures. In addition, 32P emits only xcex2-radiation with an excellent depth penetration in tissue of approximately 6 mm. Unlike many other radionuclides under consideration for targeted radiotherapy, it is not inherently toxic, and is currently used clinically in some non-targeted applications, for example, for the treatment of ovarian cancer and polycythemia rubra vera.
Another radioisotope of phosphorus, 33P, has received even less attention than 32P. 33P shares the same chemical properties as 32P, and has similarly desirable radiochemical characteristics. It is available in high specific activity, and has a 25-day half life with a xcex2-particle emission energy of 0.25 MeV, approximately 15% of the value of the xcex2-emission energy of 32P.
One reason radioactive phosphorus has received relatively little attention for targeted radiotherapy applications has been the difficulty of linking it to targeting proteins. Most of the methods currently known are non-specific and slow, and do not efficiently incorporate radionuclide into the targeting protein.
Another reason that radioactive phosphorus has received relatively little attention for radioimmunotherapy, is the rapid dephosphorylation of 32P in human serum. It has been proposed that enhanced serum stability of 32P in, e.g., the kemptide sequence can be achieved by modifying the primary sequence of receptor peptides.
One very general method of labeling proteins with 32P is simply to incubate the protein with xcex1-32P-labeled nucleoside triphosphates. Schmidt et al., FEBS Lett. 194:305 (1986). The mechanism for the labeling reaction is unknown. The method is slow and gives only poor incorporation of label (less than 1% of the protein molecules are labeled), and is thus too inefficient for therapeutic use.
A second general method of 32P labeling is to incubate Proteins with [xcex3-32P]ATP or H332PO4 in the Presence of chromium ions. Hwang et al., Biochim. BioPhys. Acta 882:331 (1986). This method is relatively rapid, but gives an unknown level of label incorporation and also leaves toxic chromium ions bound to the proteins, which would be therapeutically unacceptable.
A third general method is the use of 32P-diphenylphosphinothionyl chloride as a reactive labeling compound. De Boer et al., Clin. Exp. Immunol. 3:865 (1968). This reagent is thought to react non-specifically with lysine residues in proteins to form a highly stable conjugate, but approximately 50% of the radioactivity also associates non-covalently with the labeled protein. Although this method allows labeling of proteins to high specific activity, the labeling agent is only poorly water soluble, and to achieve good labeling yields large excesses of reagent must be used, wasting relatively large amounts of hazardous radioactive materials.
A less general method of 32P labeling is the use of periodate-oxidized [xcex1-32P]ATP to affinity-label proteins containing an ATP-binding site. Clertant et al., J. Biol. Chem. 257:6300 (1982). Because many targeting proteins which are of therapeutic interest, in particular antibodies, do not contain ATP-binding sites this method is therefore of little general utility.
A more recent method, intended for labeling antibodies for radiotherapy, involves the chemical conjugation of protein kinase substrate peptides to antibodies. Foxwell et al., Brit. J. Cancer 57:489 (1988). The conjugates are labeled by treatment with [xcex3-32P]ATP in the presence of the catalytic subunit of cAMP-dependent protein kinase (protein kinase A, PKA), which transfers 32P-phosphate to a serine residue in the substrate peptide. This method showed differences in the xcex2-phase half-life between the 32P-labeled antibody and a corresponding 131-I-labeled antibody, and also high 32P uptake in the bone of animals injected with the labeled antibody. Creighton et al., xe2x80x9cThe development of 32P technology for radioimmunotherapyxe2x80x9d in MONOCLONAL ANTIBODIES 2. APPLICATIONS IN CLINICAL ONCOLOGY. A.A. Epenetos, ed., Chapman and Hall, (1993) pp. 103-109. These results indicate in vivo instability of the label, presumably due to the action of protein phosphatases which are ubiquitous in eukaryotic cells.
Recently, Leung et al., Cancer Res. 55:5968s (1995) successfully expressed a hMN-14Fabxe2x80x2-kemptide fusion protein that can be enzymatically phosphorylated with 32P by bovine protein kinase. The kemptide sequence was attached at the C-terminus of the human IgG1 hinge region and phosphorylation of the sequence did not affect the immunoreactivity of the fusion antibody, thus resolving the problem of non-site-specific conjugation associated with the chemically linked kemptides.
While these methods serve well to phosphorylate the protein, the attached 32P was rapidly dephosphorylated from the kemptide sequence in human serum, probably by serum phosphatases. Although enhanced serum stability of the 32P in the kemptide sequence can be achieved by modifying the primary sequence of receptor peptides, it is always at the expense of reduced ease of phosphorylation by protein kinases. While these findings have discouraged the use of kemptide sequence as the substrate from 32P labeling, they nevertheless demonstrated the feasibility of enzymatically labeling antibodies and antibody fragments engineered with the appropriate phosphorylation peptide sequences.
pawson (U.S. Pat. No. 5,352,660) and pawson et al. (U.S. Pat. No. 5,667,980) disclose methods for assaying a medium for the presence of a substance that affects an SH2-phosphorylated ligand regulatory system. The patents also disclose a SH2-phosphorylated ligand complex which is capable of interacting with an SH2 like domain or subdomain thereof. pawson and pawson et al. fail to recognize the ability of the SH2 domain to inhibit in vivo dephosphorylation.
It is apparent therefore, that 32P-and 33P-labeling proteins are greatly desired for in vivo therapeutic and diagnostic usage. In particular, substances wherein the 32P or 33P label is stable in vivo, are not readily dephosphorylated, and which do not compromise the binding abilities of these proteins are needed.
A radiotherapeutic agent precursor comprising a protein selected from the group consisting of:
X-Y-L-SH2 xe2x80x83xe2x80x83(I); 
X-SH2-L-Y xe2x80x83xe2x80x83(II); 
Y-X-SH2 xe2x80x83xe2x80x83(III);
and 
wherein X is a targeting peptide, Y is a phosphorylation peptide, L is a flexible linker and SH2 represents an SH2 domain. The invention also relates to 32P or 33P radiolabeled agents of formulae (Ixe2x80x2)-(IVxe2x80x2):
X-Yxe2x80x2-L-SH2xe2x80x83xe2x80x83(Ixe2x80x2); 
X-SH2-L-Yxe2x80x2xe2x80x83xe2x80x83(II); 
Y-X-SH2xe2x80x83xe2x80x83(III);
and 
wherein X is a targeting peptide, Yxe2x80x2 is a phosphorylation peptide, L is a flexible linker and SH2 represents an SH2 domain; wherein said phosphorylation peptide is radiolabeled with 32P or 33P.